ELISA-AtoZ E
ELISA-AtoZ E
ELISA-AtoZ E
Contents.
I. ELISA is an immunoassay method 1
II. What is ELISA? 8
III. Standard curve of ELISA 12
IV. Procedure of ELISA ...Step by Step 21
V. Fundamental techniques for performing ELISA 25
VI. How to calculate ELISA assay value by EXCEL 39
VII. Important points in performing ELISA 46
VIII. Trouble shooting in ELISA 52
What is immunoassay?
The term “immunoassay” is a combined term of “immuno” (= immunological, practically
immunochemical antigen-antibody-reaction) and “assay” (= determination of the purity of a
substance or the amount of any particular constituent of a mixture according to Dorland
Medical Dictionary). So, immunoassay means a method to measure any particular substance
in a mixture using its specific-binding antibody. One of the merits of immunoassay is that we
can measure a substance that is present in a mixture of various contaminants, for example,
one constituent of blood without any purification process.
1
Important components in immunoassay
Antibody (antiserum)
In immunoasay, we use antibody as a binding reagent of high specificity and high binding
ability, that is, high affinity to the substance to be measured.
Antibody against a particular substance is obtained by immunizing an animal (e.g. rabbit)
with the substance. Some substances easily cause antibody production with a minute amount,
while others cannot produce antibodies easily, and need the help of an adjuvant like Freund’s
complete adjuvant that helps immune response.
Antigen and hapten
We call substances that can produce antibody and can bind the antibody “antigens”. Those
antigens have generally large molecular sizes over 1000 Dalton. Substances with smaller
molecular sizes cannot produce antibody by itself, but they can bind antibody if produced.
Those substances are called “haptens”. In order to get antibody for hapten, it has to be coupled
with some carrier proteins of large molecular size. We can get antibody by immunizing an
animal with such “immunogen” Antibody appears after the first innoculation is IgM-type
which is a pentamer of the basic component of antibody. This type of antibody changes to
IgG-type after the repeated innoculations. This is called “class switch”, and mostly IgG type of
antibodies are used in immunoassay.
Antibodies are also called “immunoglobulins” from their chemical nature, and the basic
molecule is composed of two heavy (H) chains and two light (L) chains, molecular weighs are
50,000-70,000, and 23,000, respectively. Immunoglobulins are classified from the structure of
H-chain as IgG, IgM, IgA, IgD and IgE. L-chains are classified into 2 types,
lambda and kappa. IgG, IgD, and IgE are of basic structure and molecular weights are
150kDa, 170-200 kDa, and 190 kDa, respectively. IgA is a dimer type with the molecular
weight of 390 kDa, while IgM is a pentamer type of 900 kDa.
A certain area located at one end of H-chain and L-chain is called variable region (VH, VL)
B B B B
because of the variability in amino acid sequences within the same class, while the rest of the
chain is called constant region (CH, CL). The variable regions are though to be the place for
B B B B
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culture is successful, the single clone of hybridoma will produce an antibody that recognizes
single epitope, and we can get enough monoclonal antibody by trasplantation of such single
clone to mouse abdominal cavity. Because a monoclonal antibody recognizes only one place of
the antigen, by selecting two monoclonal antibodies, we can easily capture an antigen at two
epitopes located at the places we want, for example, at A-chain and B-chain of insulin.
Affinity of an antibody to the corresponding antigen is expressed by association constant
(Ka) or dissociation constant (Kd).
When angen and antibody are mixed, they will bind to form antigen-antibody complex.
Ag + Ab ⇔ Ag-Ab
This reaction is reversible, and antigen-antibody complex will separate to each component.
Association constant is defined as:
Ka = [Ag-Ab] / [Ag][Ab]
Where [Ag-Ab], [Ag], [Ab] are concentrations of components expressed in M at
the equilibrium state. Kd is defined as 1/Ka
Association constant of antigen-antibody reaction seems to be very large, as shown in the
table below.
(M)
Antibody
Anti-ovine LH 1.5 x 1011 P 6.6 x 10−12 P
P
Anti-insulin 5 x 1011 P
P 2 x 10−12
P
P
Receptor (R)
Estrogen R 1010
P
P 10−10
P
P
Testicular FSH-R
Rat 1.5 x 108 P
P 6.7 x 10−9 P
P
sparrow
Japanese quail 2.4x108 P
P 4.1 x 10−9 P
P
Binding protein
cAMP binding protein 5 x 108 P
P 2 x 10−9 P
P
(Here, antibodies are polyclonal. Affinity of monoclonal antibody is told to be less than polyclonal antibody.)
However, if concentrations of both antigen and antibody are very low, for example both of
them are as low as 1 pM, the binding rate will be only 38% even if association constant is as
high as 1012 M-1. (If molecular weight of antigen is 30,000, 1pM is 30pg/ml) as shown the
P
P
P
P
figure below. When we think about blood levels of hormones (around 1-10ng/ml), binding
reaction will occur at such concentration, and we can understand that the affinity of antibody
3
is a very important factor.
60
50
40 38.2
30
20
0.98 4.55
10 8.39
0
0.01 0.1 1 10 100 1000
Antigen concentration = Antibody concentration, pM
Standard preparation
Standard preparation is necessary for immunoassay. Using a standard preparation, we
draw a standard curve from graded reaction results of various standard concentrations, and
by comparison of a sample reaction result with the standard curve, we get assay value of the
sample. Assay results are expressed by either absolute amount of the target substance, such
as weight and concentration, or comparative amount such as potency (biological unit,
enzymatic activity, and officially defined international unit).
Even if the absolute amount of a substance is required, purity of the standard preparation
is not necessarily requested if the assay results are not expressed by the amount of the
standard preparation employed. If the purity of the standard preparation, i.e., the content of
the pure substance in the standard preparation is known, it is enough to be used in assay
because assay results can be expressed in terms of pure substance. Sometimes, highly pure
preparation is unstable and easily denatured or inactivated, and sometimes might be lost by
adsorption to the wall of a container. In such case, some protective substances are
indispensable.
Very often, an international organization, like WHO, issues standard preparations in which
the amount of the substance to be measured is expressed as International Unit (IU) per vial.
This IU does not necessarily express the amount pure substance, and in many cases IU is
defined, for example, from the biological potency. So, if this kind of preparation is used, assay
results will be expressed like IU/ml, IU/mg, etc.
If a target substance has small molecular size and highly purified preparation is easily
obtained and is stable, it would be easy to use such pure substance as a standard preparation,
and assay value is expressed like ng/ml, ng/mg, etc.
4
Labeling materials
In immunoassay, it is necessary to use any marker to know the antigen-antibody
binding. For such purpose, we label either antigen or antibody with some materials that
do not interefere with the binding. We use radioisotopes, enzyme, fluorescent substance
like FITC, chelates of lanthanide elements, and so on, as labeling materials. These are
also very important factors in setting up immunoassays.
5
Quantitative aspects of the reaction between insulin and insulin-binding antibody.
Berson, S. A. and Yalow, R. S.
J. Clin. Invest. 38, 1996-2016, 1959
In the presence of fixed amounts of anti-insulin antiserum and iodine-131 labeled insulin,
non-radioactive insulin added to this system binds anti-insulin antibody in competition with
the labeled insulin if the amount of the antibody is relatively small enough. In the absence of
non-radioactive insulin, the amount of radioactive insulin bound to antibody, B/F is maximal
where B and F are radioactivity of bound and free insulin, respectively. If the amount of
non-radioactive insulin increased, the specific radioactivity of the insulin mixture (radioactive
and non-radioactive) is decreased by isotope dilution. So, if the amount of antibody-bound
insulin remains unchanged, B/F would show a hyperbolic curve. Very often B/Bo (Bo means
radioactivity in the absence of non-radioactive insulin) is also used instead of B/F. Using this
expression the standard curve will be expressed as Y = A / (X+A) x (b/bo)
A: concentration of radioactive insulin (or radioactive ligand)
X: concentration of non-radioactive insulin (or non-radioactive ligand)
bo: concentration of antibody bound insulin (or ligand) when X = 0
b : concentration of antibody bound insulin (or ligand) when X ǂ 0
Because the antigen-antibody binding is a reversible reaction and the amount of antigen
bound to a fixed amount of antibody increases with increasing amount of antigen, b/bo will
increase if X increases, and the extent of increase is inversely related to Ka. This member
b/bo should be called “binding-increasing effect”, on the other hand A / (A+X) will decrease if X
increases, and this member should be called “isotope dilution effect”. The sensitivity of the
standard curve always worse than the curve Y = A / (A+X) due to b/bo, and the shape of the
standard curve apparently looks like hyperbolic but strictly, not.
The principle of RIA is called “competitive binding” which is the first principle used in
immunoassays. The assay methods using competitive binding principle are called
“competitive assays”, and those methods in which radioisotope are used are called
“competitive radioassays”.
In competitive assays, association constant Ka influences b/bo (larger Ka causes lower b/bo,
to make sensitivity better), and the amount of antibody used in the assay also influences b/bo
(larger amount of antibody increases b/bo causing bad sensitivity). Amount of labeled antigen
influences A/(A+X), and large amount of labeled antigen minimize isotope dilution effect,
moving the standard curve to the right.
The sensitivity of insulin radioimmunoassay was enough to measure circulating insulin
levels. So, many scientists started to apply this method to various other hormones. In RIA,
highly purified antigen is necessary for antiserum production and for radioiodination. RIA
systems in 1960’s were established mostly for hormones of animals such as sheep, pig and bull.
Use of antibody caused some difficult problems because of the strict specificity of antibody,
that is, species specificity. One RIA system established for a hormone of one species of animal,
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in many cases, cannot be applied to the same hormone of other animal species. It took some
years until most human hormones came to be measured by RIA. Significance of measurement
of human hormones in blood has been so great both in clinical diagnosis and clarification of
endocrine physiology. Yalow was awarded the Novel prize in 1977.
Development of immunoassay
Since the establishment of radioimmunoassay, other assay methods have been searched for
by changing the components of RIA, namely assay principle, binding reagent, and labeling
material.
It has been very difficult to find alternative binding reagents, and as the results of such
efforts, RRA (radioreceptor assay) and CPBA (competitive protein binding assay) were
established where only antibody has been replaced for hormonal receptor and binding protein,
while assay principle and labeling material remained unchanged. But affinity of these
reagents for target substances were proved to be not enough (see those data shown in the
table in antibody section), and good sensitivity was not obtained. So, they failed to be popular
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assay methods. RRA, however, was found to be useful in analyzing hormone receptors.
Next efforts were finding of proper labeling materials other than radioisotope without
changing assay principle and binding agent, antibody. Radioisotope has many merits such as
simple labeling procedure, easy counting of radioactivity, and simple assay procedure,
however, it needs a special laboratory from the point of protection from radiation exposure,
though very small, and short half-life of radioisotope (125I: 60days,
P
P
131I:
P
P 8 days, 3H: 12years).
P
P
Tritium has a comparatively long half life, and ”long half life” means “low specific
radioactivity”, resulting in inferior assay sensitivity. In some countries, like Japan, regulation
for radioisotope is very strict. As the results of searching for a good labeling materials, there
found enzymes, fluorescent compounds, lanthanide elements, luminescent compounds, spin
reagents (free radicals) and so on.
A crucial defect of competitive assays is that the antigen-antibody binding has to be done at
very low concentrations of both antibody and antigen (including labeled and unlabeled
antigen). So, the sensitivity is quite limited, and also assay variation is large in lower area of
the standard curve.
Different assay principle has been also a target of researchers, and a second assay principle,
sandwich binding principle, depends upon multi-valency of high molecular size antigen. If an
antigen is large enough and has two epitopes, this antigen can be caught by two different
antibodies which bind to different epitopes, and the complex looks like sandwich, a slice of
ham (antigen) between two pieces of bread (antibody).
The assay method using this principle without changing labeling material and binding
reagent is IRMA (immunoradiometric assay), and as in the case of RIA, labeling has been
made with enzymes, fluorescent compounds, lanthanide elements, etc., as shown in the figure.
This principle has possibility to be more sensitive than competitive binding principle.
8
efficiency of capturing antigen for excellent sensitivity, antibody preparation is used as IgG
fraction, or monospecific antibody fraction obtained by an affinity chromatography rather
than crude gamma-globulin fraction prepared by ammonium sulfate fractionation.
antibody has been labeled with an enzyme such as horseradish peroxidase (HRP).
4) The enzyme-labeled second antibody will bind to the antigen which is bound to the capture
9
antibody on the bottom area of wells. This means that the enzyme (HRP) is also fixed on the
bottom of wells. The amount of the antigen captured is proportional to fixed enzyme.
5) Enzyme activity is measured by adding a chromogenic substrate of this enzyme. In the case
of HRP, tetramethylbenzidine (TMB) is often used. After incubation for some period, the
chromogenic substrate is changed to a colored product. The reaction is stopped by adding a
reaction stopper, e.g. diluted sulfuric acid, and absorbance is measured using a plate reader.
6) The standard curve is prepared from the concentration of standard solutions and their
absorbance. And the sample assay values are obtained from the absorbance using the
standard curve (calibration curve).
Avidin: A basic glycoprotein present in raw egg white. Produced in oviducts of avians and
amphibians. It is tetramer of essentially same single chain subunit of 128 amino acids
10
(N-terminal amino acid is alanine and C-terminal glutamic acid). Molecular weight is about
66,000. It is destructed by heat treatment (cooking) and irradiation.
Avidin binds firmly with biotin and inactivates it. Each subunit can bind one biotin. The
dissociation constant, Kd, is 10-15M. Feeding of a large amount of egg white to rat and
P
P
chicken makes biotin deficient and causes skin lesion and growth retardation. This effect is
reversed by biotin administration.
Origin Horseradish
Molecular size 40,000
Optimal pH pH 6.5
Substrate Peroxides: H2O2, CH3OOH, C2H5OOHB B B B B B B B B B
11
III. Standard curve of ELISA
1. Shapes of standard curves depending on scales in X- and Y-axes.
Standard curve of ELISA prepared by plotting standard concentration on X-axis and
absorbance on Y-axis, both in normal scale, looks like a linear line except for lower
concentration area. In much higher concentration area, it becomes a curve linear with
gradually decreasing slope (not seen in this figure). But this standard curve is very
inconvenient, because the lower standard points are very close. Especially in manual reading
is impossible in this area. Here I show an example of insulin ELISA kit.
12
Dog insulin standarad curve
2.500
Abs. 450(⊿620) nm
2.000
1.500
1.000
0.500
0.000
0 5 10 15
Insulin, ng/ml
If you plot only lower concentration area, you can see the detail of the standard curve,
which is shown below. But still the lowest part is rather difficult to use for manual reading.
0.140
0.120
Abs. 450 (⊿620) nm
0.100
0.080
0.060
0.040
0.020
0.000
0 0.5 1 1.5
Insulin, ng/ml
If the scale of X axis is changed to logarithmic scale as in the figure shown below, all the
standard points become distinguish, however, manual reading is still difficult owing to the
distances in Y-axis are close in low concentration area.
2.000
Abs. 450(⊿620) nm
1.500
1.000
0.500
0.000
0.1 1 10 100
Insulin, ng/ml
13
When both X- and Y-axes are transformed to logarithmic scales, the standard curve looks
nearly linear, though still curve linear strictly. By such plotting all the standard points are
located with enough intervals with regard to both axes, and also third order regression fits
very well due to the slight curve. Manual reading is also easy.
1.000
0.100
0.010
0.001
0.1 1 10 100
Insulin, ng/ml
But in EXCEL, simple change of to logarithmic scales is not suitable for calculation of
regression, because data themselves are not transformed to logarithm in EXCEL. You can
only see how the standard curve looks like. If you try to get regression equation without
transformation, you will obtain very poor results.
-0.5
-1
-1.5
-2
-2.5
-1 -0.5 0 0.5 1 1.5
Log(insulin conc.)
In order to get a best-fit third order regression equation in EXCEL, firstly transform
standard concentration and absorbance to logarithmic value, then draw the curve. As you can
see in the above figure, you can obtain a third order regression equation with excellent fitness
to all the standard points (please, see R2 in the figure). . For calculation of sample assay
P
P
values, refer to the part “VI. How to calculate ELISA assay value by EXCEL” on page 38.
14
that antigen-antibody binding is performed in solution (not solidified system), and that
U U
antigen and antibody bind at 1 to 1, following the low of mass action as is described in former
section “Important factors in immunoasssay”. Though, in ELISA, antibody is fixed in on the
wall of wells, at least this simple postulation is useful in understand how standard curve
changes depending on basic factors, association or dissociation constant, and capture antibody
concentration. Later on, I will show what’s going on practically.
In the former section, log-log transformed standard curve looks a slight sigmoid curve. Why
is not it linear?
From the equation of antigen-antibody reaction, below,
where initial antigen concentration: H, antibody concentration: R ,
dissociation constant: Kd, and bound antigen concetration: b (unit:M),
Kd=(H-b)x(R-b)/b
b2-(Kd+H+R)b+RH=0
Kd + H + R − ( Kd + H + R )2 − 4 HR
b=
2
Above graph shows changes of bound antigen concentration to capture antibody (10000pM)
with various Kd when initial 5-1280pM antigen is added. If the results are plotted with
log-log scales, the binding goes up straightly to some points then bends.
It is shown that the standard curve move toward right depending on Kd, indicating that
assay sensitivity is related to Kd. If Kd is small (that is, affinity constant is large) the
sensitivity becomes excellent.
When the results are expressed with normal scales, like the graph below, each binding
curve shows different slopes depending on Kd.
15
Binding of Ag to fixed Ab(Fixed Ab = 10000pM)
12000
10000
Antigen bound, pM
Kd= 100
8000
Kd= 300
6000 Kd= 1000
Kd= 3000
4000 Kd=10000
2000
0
0 5000 10000 15000 20000 25000 30000
Antigen added, pM
If Kd is constant, the standard curve (binding curve) moves to the right when fixed antibody
is increased.
1000
Fix 500
100
Bound antigen
Fix 1000
Fix 2000
,pM
10 Fix 5000
Fix10000
0.1
1 10 100 1000
Added antigen, pM
In this simulation, we can understand the changes of standard curve, especially, that of
sensitivity due to Kd and capture antibody. But the lower standard concentration area is
linear, and different from the real ELISA standard curve in which lower area is curve linear.
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incubated for 2 hours. After incubation 100µl of the reaction mixtures were transferred to
plate B and incubated for 2 hours. Plate A and B were washed and treated through the rest of
the assay procedure. A standard curve was prepared using plate A, the amounts of insulin in
plate B were calculated which indicated residual insulin.
Results are shown in the following figure and table.
3.0
Standard curve
2.5
Residue
abs.450(⊿620)nm
2.0
1.5
1.0
0.5
0.0
0.1 1 10
Insulin(ng/ml)
It is shown that binding percentage decreases if added insulin is less. If 10ng/ml insulin is
added, about 90% is bound to the capture antibody, while binding percentage is about 45%
when 0.156ng/ml is added.
The figure below is obtained by plotting the logarithmic concentration of added insulin
against binding percentage. It seems linear, indicating that binding percentage is
proportional to logarithmic concentration of added insulin.
17
Added antigen and binding percentage
100
90
80
70
Binding %
60
50
40
30
20
10
0
0.1 1 10
Insulin added, ng/ml
This may be explained from the retardation of the antigen-antibody reaction in solid phase
antibody system. Solidified antibody cannot move around the solution and only antigen can
move to fixed antibody. If the concentration of antigen is
low, the efficiency of finding the partner and binding would
be decreased, and takes more time. Incubation for 2 hours
may not enough for the low population of antigen.
The author calls this effect “binding decreasing factor
1(BDF-1)”.
In ELISA, we have to take another factor into
consideration. It is the binding of labeled antibody to the
antigen captured on solidified antibody. If the all the
captured antigen molecules bind the enzyme labeled second
antibody the specific enzyme activity (enzyme
activity/bound antigen) must be constant. The author found
that the specific enzyme activity that was expressed by absorbance/bound insulin was not
constant but also proportional to added insulin concentration. The author calls this factor
“binding decreasing factor 2 (BDF-2)”.
In order to simulate the ELISA standard curve composed of antigen concentration and
absorbance, bound antigen calculated from the antigen-antibody reaction in liquid phase
should be multiplied by BDF-1 and BDF-2 for correction.
The author tried the simulation for rat insulin ELISA Kit by estimation of bound insulin in
liquid phase with postulation of solid antibody concentration of 10000pM and dissociation
constant of 1000pM, and standard insulin 0.156-10ng/ml (27-1724pM). Then using the
equations of BDF-1 and BDF-2. The results obtained are shown in the figure below.
The simulated standard curve was compared with the real standard curve.
Both curves coincided very well.
18
Simulated and observed standard curves
10
1
Simulated
Abs.
Observed
0.1
0.01
0.156 0.313 0.625 1.25 2.5 5 10
Insulin concentration,ng/ml
1 2 3 4 5 6 7 8 9 10 Mean SD CV%
2.398 2.484 2.492 2.464 2.511 2.513 2.486 2.505 2.5 2.492 2.4845 0.0336 1.3545
1.286 1.271 1.272 1.263 1.279 1.3 1.299 1.306 1.285 1.323 1.2884 0.0184 1.4349
0.61 0.604 0.609 0.612 0.61 0.602 0.597 0.606 0.602 0.624 0.6076 0.0073 1.2173
0.275 0.292 0.283 0.286 0.284 0.284 0.271 0.29 0.273 0.287 0.2825 0.0071 2.5375
0.147 0.147 0.144 0.152 0.146 0.149 0.146 0.146 0.149 0.148 0.1474 0.0022 1.5068
0.086 0.088 0.091 0.091 0.092 0.092 0.088 0.089 0.089 0.092 0.0898 0.0021 2.3358
0.07 0.071 0.07 0.075 0.069 0.071 0.071 0.07 0.07 0.072 0.0709 0.0016 2.3460
0.06 0.064 0.06 0.061 0.06 0.06 0.061 0.061 0.061 0.057 0.0605 0.0017 2.8362
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SD and CV of Absorbance (10Replicates assay)
0.04 3
0.035
2.5
0.03
SD of absorbance
2
0.025
CV, %
0.02 1.5
0.015 SD 1
0.01 CV%
0.5
0.005
0 0
2.48 1.29 0.61 0.28 0.15 0.09 0.07 0.06
Absorbance of each Std point
In this figure, data are arranged from the largest standard concentration to the least.
The absolute values of SD of standard points increase in parallel with the standard
concentration, however, CV (RSD) shows an opposite tendency, i.e. CV at the least standard
point is about twice of that at the largest point.
Increase of CV in lower concentration area is possibly influenced by variation of a
plate-reader, heterogeneity of well-plate (such as thickness of the bottom and flaw) and
non-specific adsorption of labeled antibody.
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Change of absorbance by 2% will cause nearly 2% change around 0.5 to 2.5 ng/ml (in the
middle area of the standard curve), and shows a tendency to increase outside this area. The
lowest and the highest standard show CV nearly 4%. This is due to the difference in slope of
the standard curve. But from our experience, influence of variation in absorption is far
smaller than that of competitive assay, especially in lower concentration area. The slope of
ELISA standard curve expressed by bi-logarithmic scale is steeper than that of competitive
assay.
21
○ Horseradish peroxidase (HRP)-second antibody conjugates (D) is provided as an
concentrated solution, and is used after dilution with the buffer (C), in most cases, 1:100..
○Concentrated washing buffer (I) is used after dilution with purified water to 1:10.
○Chromogenic substrate solution (F) and reaction stopper (H) are used as they are.
All the reagent solutions should be used after getting back to room temperature (20-25C).
Assay procedure
Remove the cover sheet of the microplate after getting back to room
temperature.
(1) Rinse the anti-CRP coated wells (A) by filling the washing buffer and discard 3 times,
then strike the plate upside-down onto folded several sheets of paper towel, and
remove the excess buffer.
(2) Pipette 50µl of sample solution to the wells for samples.
(3) Pipette 50µl of the standard solution to the wells for preparing standard curve.
(4) Shake the plate gently on a plate shaker for 10-15 seconds.
(5) Incubate for 1 hour at room temperature (20-25C).
(6) Discard the reaction mixture, and then wash wells as described in (1).
(7) Pipette 50µl of peroxidase-conjugated anti-CRP solution to all wells. Then shake
gently on a plate shaker for 10-15 seconds.
(8) Incubate the plate for 1 hour at room temperature.
(9) Discard the reaction mixture, and then wash the plate as (1).
(10) Pipette 50µl of chromogenic substrate solution to wells, and shake as (4).
(11) Let the plate stand for 30 minutes at room temperature.
(12) Add 50 µl of the reaction stopper (H) to all wells and shake.
(13) Measure the absorbance of each well at 450 nm (sub-wave length, 620nm) by a plate
reader within 30 minutes.
Summary of Assay Procedure
Antibody-coated 96 well microplate
↓Washing 3 times
Sample or Standard 50µl
↓Shaking and reaction for 1 hour at room temp.
↓Washing 3 times
Peroxidase-conjugated anti-CRP 50µl
↓Shaking, and reaction for 1 hr. at room temp
↓Washing 3 times
Chromogenic substrate solution 50µl
↓Shaking, and reaction for 30 mins. at room temp
Reaction stopper 1M H2SO4
B B B B 50µl
↓
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Shaking and measurement of absorbance
at 450nm(sub. 620nm)
Room temp.: 20~25C
2. An example of kits with biotin-labeled second antibody and HRP-labeled avidin.
...Mouse leptin ELISA KIT
Components of the kit
Reagents Amounts
(A) Anti-leptin-coated microplate 96 wells(8x12) / 1 plate
(B) Standard mouse leptin solution (5,000pg/ml) 500µl / 1 vial
(C) Buffer solution 60ml/1 vial
(D) Biotin-conjugated anti-leptin 200µl/ 1 vial
(E) Peroxidase-conjugated streptavidin 200µl/ 1 vial
(F) Chromogenic substrate reagent (TMB) 12ml/ 1 vial
(H) Reaction stopper (1M H2SO4) B B B B 12ml/ 1 vial
(I) Concentrated washing buffer (10x) 100ml/ 1 bottle
○Standard solution (B) is provided as an original concentrated solution. A series of
standard solutions of various concentrations are prepared by dilution of this original
solution with buffer (C), in most cases, by serial dilution, while others by proportional
dilution.
An example of preparing standard solutions
Std. Conc.
5000 2000 500 100 50 25 10 0
(ng/ml)
Std. sol.(µl) Orig. sol. 100** 100* 100* 200* 200* 200* 0*
Buffer (µl) 0 150 300 400 200 200 300 300
**Original standard solution, *One rank higher standard solution
○ Biotin-conjugated second antibody solution (D) is provided as an concentrated solution,
and is used after dilution with the buffer (C), in most cases, 1:100..
○Horseradish peroxidase (HRP)-conjugated avidin (E) is provided as an concentrated
solution, and is used after dilution with the buffer (C), in most cases, 1:100..
○Concentrated washing buffer (I) is used after dilution with purified water to 1:10.
○Chromogenic substratee solution (F) and reaction stopper (H) are used as t hey are.
All the reagent solutions should be used after getting back to room temperature (20-25C).
Assay procedure
Remove the cover sheet of the microplate after getting back to room temperature.
(1) Rinse the anti-leptin coated wells (A) by filling the washing buffer and discard 4 times,
then strike the plate upside-down onto folded several sheets of paper towel, and remove
the excess buffer.
(2) Pipette 40µl of buffer solution into the wells for samples, then add 10µl of sample to
23
each well. Alternatively, if you use larger sample volumes (X µl), the volumes of
buffer(C) should be (50 − X) µl to adjust the final volume to 50 µl.
It would be also convenient to dilute the assay samples first in test tubes, and pipette
50µl of the diluted sample to a well.
(3) Pipette 50µl of the standard solution to the wells for preparing a standard curve.
(4) Shake the plate gently on a plate shaker.
(5) 50µl of biotin-conjugated anti-leptin solution to all wells. Then shake gently on a plate
shaker.
(6) Incubate the plate for 2 hours at room temperature.
(7) Discard the reaction mixture, and then wash the plate 4 times as described in (1), and
remove excess washing buffer remaining in the wells as (1).
(8) Pipette 100µl of HRP-conjugated avidin solution to all wells, and shake as (4).
(9) Incubate for 30 minute at room temperature.
(10) Discard the reaction mixture, and then wash the plate 4 times as (1), and remove
excess washing buffer
(11) Pipette 100µl of chromogenic substrate solution to wells, and shake as (4).
(12) Let the plate stand for 30 minutes at room temperature.
(13) Add 100 µl of the reaction stopper (H) to all wells and shake.
(14) Measure the absorbance of each well at 450 nm (sub-wave length, 620nm) by a plate
reader within 30 minutes.
Summary of Assay Procedure
Antibody-coated 96 well microplate
↓Washing 4 times
Sample* or Standard 50µl
↓
Biotin-conjugated anti-leptin antibody 50µl
↓Shaking and reaction for 2 hour at 20~25C
↓Washing 4 times
Peroxidase-conjugated avidin 100µl
↓Shaking, and reaction for 30 min at 20~25C
↓Washing 4 times
Chromogenic substrate solution 100µl
↓Shaking, and reaction for 30 min. at 20~25C
Reaction stopper 1M H2SO4
B B B B 100µl
↓
Shaking and measurement of absorbance
at 450nm(sub. 620nm)
24
V. Fundamental techniques for performing ELISA
○ When taking out the pipette, touch the inner wall of the container of the sample solution
with the tip to remove excess solution outside of the tip (touch and go).
○ When discharge
the solution to a
well, push down
the plunger to the
end to deliver the
solution
completely (blow
out), then touch
and go.
○ Exchange the tip
for the next new
sampling.
○ In case to deliver
the same solution
to several wells in the same volume, you can use the same tip without pre-wetting from the
second well.
b. “Co-washing” method
This technique can be used only in the case when some buffer or solution is already in the
25
well. If there is no liquid in the well, use pre-wetting method.
○ Set a new tip, and pushdown the plunger to the first stop, and fill up the solution to be
delivered.
○ Take out the pipette after “touch and go” at the inner wall of the container.
○ Dip the top of the tip in the buffer of the well and deliver the solution to the well by
pushing the plunger to the first stop, then within the range of the first stop, repeat filling
and discharging of the liquid in the well 2 or 3 times (co-washing), and finally blowout the
solution in the tip by pushing down the plunger to the end.
○ Take out the pipette after touch and go. Then exchange the tip.
These methods are described according to the instruction paper for Eppendorf pipette.
● Pipetting should be carried out using one of the methods described above.
But do not mix up these two methods.
● If possible, we recommend “Co-washing” method for serum and plasma samples because of
high protein concentration with considerable viscosity.
● “Co-washing” method is also recommended for small sample volumes less than 10mcl.
● When sampling volume is rather large, like 50-100mcl, “Pre-wetting” method would be
suitable.
● Please, pipette every sample and reagent solution after they return to room temperature.
Operation under higher or lower temperature than room temperature may cause inaccurate
pipetting, and increase assay variation.
● Variation of temperature of the reaction mixtures would also influence reaction velocity.
● Generally, after freezing and thawing, solutes would be concentrated to the bottom part of
the liquid. So, any frozen samples should be stirred by Vortex-type mixer and made
homogeneous after thawing.
26
b. Proportional dilution
Prepare small test tubes as many as the standard solutions including zero point, and write
numbers on them.
Add the indicated volumes of buffer to the tubes. Generally, the volumes are different from
tube to tube. So, take enough care.
Add indicated volumes of the original standard solution to the tubes, and mix well.
Tube No. O should contain only buffer. In some cases, the original standard solution itself is
used as the highest standard
27
out the volume as indicated in the instruction paper, and add it to the vessel, and mix well.
4. Dilution of concentrated washing buffer
To a 1000ml beaker, add all the content of the bottle containing concentrated washing
buffer, then add a little less than 900 ml of purified water.
Make the mixture to 1000ml by adding purified water little by little. Then mix well.
Transfer this washing buffer to a plate washer, or to a 500ml washing bottle with a jet
nozzle.
5. Structure of antibody-coated microplate and treatment
28
a. Before assay, fill the wells with washing buffer using a washing bottle with a nozzle. Then
shake off the buffer from the plate onto a sink. Repeat filling and shaking off as many
times as indicated in the instruction paper.
Complete Removal of residual liquid after washing and pouring off.
After final shaking off the buffer from the plate, strike the plate on to some sheets of paper
towel for several times to remove the residual buffer.
After confirming that no liquid remains in the wells, start pipetting of the reagent solution
of the next step.
b. Washing after the first reaction: The first washing after the first reaction with standards or
samples: Shake off the reaction mixture from the plate onto a sink.
Then add washing solution using a multi-delivery pipette set at 250mcl to avoid carry-over
caused by flowing out the buffer to other wells. Then shake off the buffer. From this step on, it
is not necessary to be afraid of carry-over.
Add new washing buffer from the washing bottle with nozzle, and shake off.
Repeat filling and shaking off as many times as indicated. Then completely remove residual
washing buffer as shown above.
c. Washing after other reaction: Shake off the reaction mixture from the plate into a sink.
Add new washing buffer from the nozzled washing bottle, and shake off.
Repeat filling and shaking off as many times as indicated. Then completely remove residual
washing buffer as shown above.
29
For pipetting reagent solutions common to all the wells, a multi-delivery type pipette (e.g.
Eppendorf multipette plus) is suitable.
We should be careful in using this type of pipette;
○After filling of the liquid, remove the air, and deliver the first one or two strokes back to the
container.
○When deliver the liquid to wells, avoid too strong strokes. A strong stroke may cause
splashing out of the liquid.
○ After delivery of the liquid to a well, perform “touch and go” to the wall of the well.
○Do not dip the top of the tip in the well.
After addition of a reagent solution, the microplate should be shaken to mix the solutions. A
short shaking is enough because of the small volume of the reaction mixture.
We recommend to use a microplate mixer (microplate shaker) as shown above. Set the plate
on it, and shake at 800 rpm for approx. 10 seconds. Repeat 3 times.
30
If a microplate shaker is not available, shake the plate by hand as is described below.
Place the plate on the flat and smooth surface of a laboratory table, hold the plate and move
the plate roundly to draw circles rapidly for approx. 10 seconds while lightly pressing the
plate on the surface. Repeat 3 times.
9. Color generation
Bluish color generation will occur during incubation with TMB solution owing to enzyme
action of HRP.
31
results are automatically calculated by proper setting.
For calculation of assay results with EXCEL, refer to “VI. How to calculate ELISA assay
value by Excel” in page 36.
13. Assay validation and validation tests for performance of an ELISA kit
Any report using an ELISA system should provide the validation data to show evidences
that the assay system gives reliable data. So, the validation tests are necessary to establish
and apply an ELISA system.
In immunoassay system there are 3 kinds of errors, namely accidental error (random error),
systematic error, and gross error (mistake).
○Accidental or random error means, in other expression, variation of assay results. This is
expressed by standard deviation, or relative standard error (RSD, in other words coefficient of
variation (CV).
RSD = CV = SD/mean x 100 (%)
○Systematic error means difference from true value or bias which is related to accuracy.
○Gross error is, in other word, mistake. This kind of error derives from careless mistake in
doing assay, troubles of equipments, denaturation of reagents, etc., which causes cancellation
of assay.
ICH (International Conference of Harmonization, Japan, U.S. and EU) issued a guideline
for immunoassay validation. Governments of above countries urged kit makers and users to
follow this guideline.
Important items for ELISA are as follows. In this text the author will try to explain in later
sections.
For standard curve
○Detection limit ( relating assay sensitivity)
○Quantitation limit ( relating assay sensitivity)
○Linearity (not requested for immunoassay)
○Range (= assay range covered by standard curve)
For random error
○Precision
Repeatability (= intra-assay precision)
Intermediate precision (= inter-assay precision in one laboratory)
Reproducibility (= Variation among different assay laboratories)
For systematic error
○Specificity
○Accuracy
For assay system
○Robustness (= stability of assay system under various conditions)
32
a. Standard curve and assay sensitivity
Assay sensitivity is defined as follows.
We can think that the sensitivity is the standard concentration to give significantly higher
absorbance than that of blank.
Another way to estimate the sensitivity is to show the concentration of the standard at the
middle point of standard curve. From this value we can guess the practical assay range.
As for lower limit of assay range, we should start from QL rather than DL because at DL,
assay precision (CV) will be 30% and is thought to be too big. At QL, CV will be 10% and
would be permissible.
The highest limit of assay range has some problems. Some people define the upper limit of
assay range to be the concentration which gives an absorbance of highest absorbance –3SD.
I myself think that absorbance should not be larger than 2.5. We cannot trust a colorimeter
if the absorbance is more. The slope of ELISA standard curve, if standard concentration goes
up, gradually becomes smaller and smaller until reaching its maximum. If slope becomes
smaller, the CV of the calculated assay value increases, so, only a minor change of absorbance
causes a big change of calculated concentration.
b. Assay precision (“Repeatability” according to ICH definition)
This is also called within assay variation or intra-assay variation. If the variation is small,
the mean assay value will be trustworthy.
This is expressed by CV % (coefficient of variation) of a sample which is measured by in one
33
assay trial. One assay sample is measured using several wells, and the mean and standard
deviation (SD) are calculated, then CV% is obtained as CV (%) = SD/mean x 100. Usually,
several samples are measured and their average CV % is shown. An ELISA system is
expected to give CV% less than 5% from our experience.
An example of within assay variation [Rat C-peptide ELISA KIT (U-type)]
Two samples, A and B were measured using 8 wells for each sample.
Well/Sample A B
1 1015 214
2 1027 222
3 1038 211
4 1043 219
5 1029 232
6 1034 209
7 1039 231
8 1041 224
mean 1033 220
SD 9.60 8.51
CV (%) 0.93 3.86
Unit: pg/ml
Rf: According to ISO 8655-5, maximum permissible random error for single-stroke dispenser
with nominal volume of 10µl is 0.1µl. If a pipette of a larger nominal volume like 50µl is used,
the maximum permissible random error is 0.2µl. Pipettes are so made. However, practically,
proficiency of pipetting technique is requested more seriously in precise delivering if the
sample is serum or plasma. They are viscous and should be pipetted very carefully. (Please,
refer to the pipette’s sections in p.25 and 52.)
34
c. Reproducibility (“Intermediate precision” according to ICH definition)
Reproducibility means that, if one sample is measured repeatedly in different assay trials,
the assay values obtained in those trials are always constant or not.
This is also called between assay variation or inter-assay variation, and is also expressed by
CV%. One sample is measured in several assay trials, and from assay values of trials mean
assay value and SD, CV (%) is calculated. If the number of wells in each trial is larger, the
more trustable reproducibility will be given. We expect CV% less than 10%, hopefully less
than 5%.
An example of between assay variation [Rat C-peptide ELISA KIT (U type)]
Three samples, C, D, and E were measured using 4 wells/sample, and assays were
repeated 4 times on different days.
35
on this problem is carried out using several candidate substances suspected to cross-react to
the system from similarity of their chemical structure to the aimed substance. Varied
amounts of candidate substances are measured, and the assay values are examined. If those
candidates give no detectable assay values, specificity of the assay system will be confirmed.
If any candidate gives a significant assay value, its cross-reactivity is expressed as the
percentage of the assay value to the amount of the candidate.
Sometimes, the apparent cross-reactivity is due to the contamination of the aimed
substance in the candidate preparation
An example of cross-reactivity test [Rat C-peptide ELISA KIT (U type)]]
Species Substance Cross reactivity(%)*
Rat C-peptide 100
Insulin Less than sensitivity
Pro-Insulin Less than sensitivity
Mouse C-peptide 75
Insulin Less than sensitivity
Human C-peptide 85
Insulin Less than sensitivity
Pro-Insulin Less than sensitivity
Pig Insulin Less than sensitivity
Cow Insulin Less than sensitivity
*Cross-reactivity was estimated with 15,000 pg/ml of the substance
e. Accuracy
Accuracy of assay system, i.e. whether the assay value obtained really reflects the amount
of the aimed substance or not, should be confirmed through several tests, like recovery test,
linearity of dilution test, and comparison with other assay system.
○ Spike recovery test
Sample serum/plasma is divided into two portions. And a certain amount of the standard
preparation is added to one portion (sometimes called “spike”), while the other is left intact.
Both portions are assayed, and from these two assay values, recovery of the standard
preparation added (spike-recovery) was estimated by subtraction. Recovery of the added
standard should be around 100% within the range of assay precision. In the recovery tests
shown below, three different amounts of the standard are added
36
110 291 111 101
161 332 152 94.5
209 379 199 95.1
Dilution test
2500
2000
Concentration, pg/ml
1500
R2 = 1.00
1000
500
R2 = 1.00
0
0 0.2 0.4 0.6 0.8 1
37
question. In trying this test, we have to collect many samples containing various amounts of
substance to be measured in a wide range. Those samples are assayed by two assay systems
for comparison. From assay values, their correlation coefficient and an equation of first order
regression (y = ax+ b) are calculated. If the standard preparation is commonly used in two
assay systems, the slope, a, must be nearly 1.0, and b must be small enough compared with
assay values. If those two systems use different preparations of the standard, the slope, a, is
not necessarily 1.0, depending on the difference of purity of the preparations. The correlation
coefficient should be nearly 1.0.
An example of comparison between Shibayagi’s two rat insulin assay kits is shown.
2100
y = 0.96x + 64
Assay value by U-type (pg/ml)
1600
1100
600
600 1100 1600 2100
Another example is the comparison of rat insulin ELISA KIT (T-type) with S-type
T-type kit cross-reacts proinsulin, while S-type is more specific to insulin, and this
difference possibly makes the slope value 1.2.
7
y = 1.2091x + 0.1058
Insulin byT-type kit (ng/ml)
6 2
R = 0.9893
5
4
3
2
1
0
0 2 4
Insulin by S-type kit (ng/ml)
38
disease (D) is known to show significant increase or decrease of the blood level of a substance,
and an ELISA system is established for this substance, this ELISA system is tested in clinical
sections as follows.
Blood samples are collected from normal subjects, D-patients, patients of other D-like
diseases to be distinguished from D, and measured for the substance in question. From the
assay data, two indices, sensitivity and specificity (though names are same to the indices for
assay performance) are calculated.
Sensitivity: percentage of significant change of the substance to disease D patients
Specificity: percentage of significant change of the substance in disease D to all signigicant
change of the substance.
If these the percentages of these two indices are high, diagnostic usefulness of the ELISA is
thought to be high.
Procedure of calculation step by step with an example of our insulin assay data
Input of data in EXCEL spread sheet.
Standard points of rat insulin: 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10.0 ng/ml
First make up a table for standard concentration and absorbance as shown below. The
example shown here is a duplicate assay, and as TMB is used as chromogenic substrate, we
measured absorbance at 450nm. If possible, as absorbance, difference of absorbance at 450nm
and 620nm is preferable. Subtract the mean of blank absorption from each mean absorbance
to make ∆Blank.
A B C D E
1 Insulin Abs.450(∆620)nm Mean ∆Blank
2 10 2.316 2.214 2.265 2.233
3 5 1.312 1.227 1.270 1.238
4 2.5 0.614 0.641 0.628 0.596
39
5 1 0.217 0.209 0.213 0.181
6 0.5 0.112 0.108 0.110 0.078
7 0.25 0.064 0.061 0.063 0.031
8 0.1 0.045 0.044 0.045 0.013
9 0 0.031 0.032 0.032
40
Click” column” as date series
5.Click “series” and choose data area of X.
6.Indicate the column of Ln(⊿Blank) (in our example G2-G8) as date area of X, then “next.”
7.Click “Title and label” tag, and write the graph title, names of X and Y axis. Click “legend”
tag and uncheck “show legend”
8.Click ”finish to show a graph on the data sheet.
3
2
1
Ln(conc)
0
-5 -4 -3 -2 -1 -1 0 1 2
-2
-3
Ln(Abs.⊿Blank)
As the graph appears as above is not good looking, so we should move X and Y axis.
To move X axis, double click the figure on X-axisto show X-axis setting window, and at
“scale” tag uncheck the automatic checking of “intersection with Y, and write the intersection
wanted (in our example, -5”, then OK. Y-axis is move in the similar way to X-axis. Wite “-3” in
our case.
Then the graph appears as shown below.
3
2
1
Ln(conc)
0
-1
-2
-3
-5 -4 -3 -2 -1 0 1 2
Ln(Abs.⊿Blank)
41
Now, we get the reverse standard curve with equation and R2. From R2, we can estimate P
P
3
3 2
y = 0.0222x + 0.1324x + 0.997x + 1.4003
2 2
R = 0.9998
1
Ln(conc.)
0
-1
-2
-3
-5 -4 -3 -2 -1 0 1 2
Ln(Abs.⊿Blank)
The R2 value, 0.9998, obtained indicates that the fitness of the third order regression
P
P
Let’s check fitness of the equation by calculation the assay value from standard data.
Using the equation we can calculate the assay value of samples and also fitness of the
regression equation to the standard curve. Before calculation of sample assay values, I
recommend to check the fitness.
In order to examine fitness, we add two columns next to the column Ln(∆Blk) (columns H
and I). For column H, we will fill calculated logarithmic concentrations, i.e. Cal.Ln(conc),
then we tranform them to normal value, and write them to the column I as Calc.conc. We
will explain the step using our example.
To the cell H2, copy a part of the third order equation shown in graph, and change x to G2
like;
=0.02*G2^3‐0.1156*G2^2+0.9754*G2+1.3934
and push Enter key. Then 2.298192 will be written into H2 cell. The again point H2 to
activate H2, and drag the fill handle of the cell until H8 to calculate all the standard data.
The next step is to write “=EXP(H2)” to the I2, and push Enter key. Then the value 9.956164
will be written in I2. After activation of the cell I2, drag the fill handle until I8. Then all the
logarithmic concentration will be transformed into normal value.
You can compare those value in the column I with those in column A, you can examine the
fitness.
The results of our example calculation are shown in the table below.
42
A B C D E F G H I
Cal. Cal.Conc.
1 Insulin Ln(conc) Abs.450(⊿620)nm mean. ⊿Blank Ln(⊿Blk) Ln(conc) Fitness
No. Abs.1 Abs.2 LN(Abs1) LN(Abs2) LN(AV)1 LN(AV)2 AV1 AV2 Mean SD CV
1 0.125 0.127 -2.07944 -2.06357 -0.30001 -0.28836 0.740811 0.749495 0.745153 0.006141 0.824131
2 0.138 0.136 -1.9805 -1.9951 -0.22739 -0.2381 0.796608 0.78812 0.792364 0.006002 0.757479
3 0.075 0.077 -2.59027 -2.56395 -0.67968 -0.65976 0.506778 0.516974 0.511876 0.00721 1.408496
4 0.096 0.093 -2.34341 -2.37516 -0.49469 -0.51827 0.609763 0.595548 0.602655 0.010052 1.667886
5 0.186 0.191 -1.68201 -1.65548 -0.00772 0.01192 0.992305 1.011992 1.002148 0.013921 1.389083
6 0.156 0.162 -1.8579 -1.82016 -0.13738 -0.10963 0.87164 0.896166 0.883903 0.017343 1.962064
7 0.256 0.251 -1.36258 -1.3823 0.231465 0.216493 1.260445 1.241715 1.25108 0.013244 1.058621
8 0.897 0.889 -0.1087 -0.11766 1.293463 1.284792 3.645387 3.613915 3.629651 0.022254 0.613122
9 1.254 1.238 0.226338 0.213497 1.633 1.619408 5.119207 5.050098 5.084653 0.048868 0.961079
10 2.213 2.254 0.794349 0.812706 2.286936 2.309934 9.844731 10.07376 9.959246 0.161948 1.626109
In our example, results with 10 samples are shown. More samples can be treated.
Preparation of a template for calculation
It would be convenient to prepare a template for ELISA calculation and store the file. What
you should do is only to take out the file and fill the table with absorbance of standard and
samples, and store the results table with a new file name. Duplicate assay is intended. The
figures 1and 2 indicate well1 and well2, respectively.
43
Preparation of a template for standard curve.
Procedure
First, prepare a table in EXCEL.Let us call the cells A-1~H-8.
Cells in line 1 are used only for identification. Just write as are shown in the model table.
In the cell C2, write “=LN(A2)”
In the cell E2 write “=(C2+D2)/2
Leave F2 untouched. To the cell F2 write “=E2-average absorbance of blank” after assay.
In the cell G2 write “=LN(F2)”
Leave the H2 untouched. (After assay write the equation to H2)
In the cell I2 write “=EXP(H2)”
Then you will get a table as shown below.
A B C D E F G H I
1 Conc. Ln(conc) Ab 1 Ab 2 Mean ∆Blank Ln(∆Blank) CalLn(conc) Cal conc
2 #NUM! 0 #NUM! 1
Store the template and use a copy.
How to use the template for the standard curve
Using the data for standard solutions, first finish calculation until the column G.
Before starting calculate the average of blank absorbance.
a. In the column A write concentrations of standard solutions starting from the highest.
Then B2 cell will be filled with the logarithmic transformation of the highest standard
concentration.
b. Activate B2 cell and drag the fill handle until B8 to complete transformation.
c. Write pairs of absorbance of wells in columns C and D.
d. Point E2 to activate, and drag fill handle until E8 to obtain means.
e. Write “=E2-blank absorbance(calculated above)” in F2, then drag fill handle to F8 for
∆Blank.
f. Activate G2, then drag the fill handle until G8 to obtain logarithm of ∆Blank.
g. Get the reverse regression curve as stated above (p.32)
h. Write the equation of 3rd order regression curve in H2 as described above (p.34), then
P
P
click.
i. Activate H2, and drag the fill handle until H8.
j. Point I2, then drag I2 fill handle until I8.
k. Compare the assay values in the column I with the concentrations in A for fitness.
The equation written in H2 can be used for sample calculation, by copying.
44
A-1~N2).
Cells in line 1 are used only for identification. Just write as are shown in the model table.
Ab: absorbance of sample before subtraction of blank absorbance
∆Bk: absorbance of samples subtracted blank absorbance
LN( ): natural logarithm of ∆Bk
Cal:natural logarithm of calculated sample assay value
Av: Assay value of sample, transformed to normal number
Mean: acerage of well 1 and well 2, SD: standard deviation, CV: Coefficient of variation
Procedure
In the cells in the column A, write sample number.
The cell D2 is left empty until use. After assay write “=B2-Blank”
Blank: mean blank absorbance value
The cell E2 is left empty until use.
In the cell F2, write “=LN(D2)”
Leave the cell H2 untouched. (After assay write the regression equation)
The cell I2 is left untouched
In the cell J2, write “=EXP(H2)”
In the cell L2 write “=(J2+K2)/2
After assay fill M2 with SD using function STDEV
In the cell N2, write “=M2/L2*100”
(Do not include quotation marks in writing.)
Then you will get a template table as shown below.
Store the template until use, and use it after making a copy.
A B C D E F G H I J K L M N
1 No. Ab1 Ab2 ⊿Bk1 ⊿Bk2 LN(1) LN(2) Cal.1 Cal.2 Av.1 Av.2 Mean SD CV
2 1 #NUM! #NUM! 1 1 1 0 0
3 2
4 3
5 4
6 5
7 6
8 7
9 8
10 9
11 10
45
After preparation of this template table, store the file. After an assay, take out the file and
once store with proper naming. then input the first pair of absorbance in the cells B2 and C2,
and “=D2-average blank absorbance” in D2, and regression equation in H2, as shown in the
previous page. In this case, By dragging of the fill handle of D2 to E2, the function will be
copied to E2, and the cell numbers are changed automatically. The situation is the same with
G2, I2, and K2. The results of calculation appear in those cell of the line 2. By dragging each
fill handle down to the last cell, results of calculation will appear when each pair of sample
absorbance are input. The calculation will be completed after the input of absorbance of the
last sample.
Number of samples is not limited.
You can download the template ready to use from Shibayagi’s web site.
http://www.shibayagi.co.jp/
HTU UTH
46
Samples Hemoglobin concentration (mg/dl)
0 10 20 40
Preservatives
Antiseptics
Sodium azide (NaN3) is very often used as an antiseptic. But better to avoid this substance
B B
because it is a potent inhibitor of peroxidase from the similar reason to fluoride ion.
In ELISA, NaN3 in samples is washed out after antigen-antibody reaction, but we are not
B B
sure about complete elimination. We are afraid of influence of peroxidase inhibiting action of
NaN3 remaining in wells after washing.
B B
47
of physiological saline to make 10,000KIU/ml. The this solution is added to the blood sample
at 1/100.(v/v) (final concentration is 100KIU/ml). Then serum or plasma is separated.
Amount of aprotinin is expressed as KIU(kallikrein inhibitor unit), TIU (trypsin inhitor
unit),
BAPNA unit (N α -benzoyl DL-arginine-p-nitroanilide unit), BAEE unit
(Benzoyl-L-arginine ethylester unit), or UIP (Peptidase inhibitor unit).
1 TIU is the amount to reduce 2trypsin units by 50%.
1trypsin unit is the amount of trypsin to hydrolyses1µmole of BAPNA per minute
at 25oC, pH 7.8. P P
pH of samples
Serum or plasma, when fresh, shows pH near neutral, however, it very quickly goes to
alkaline more than pH 8 by losing CO2. Our observations on the change of pH of mouse serum
B B
Sample plasma from 2 mice were obtained by heart puncture using EDTA-2Na, and
kept standing at 2~8oC in PP vials after preparation,
P
P
7.6
7.45 7.67
P P
7.4 7.24
7.45
temperature goes up, pH will
7.2 change more quickly.
7.16
7 When frozen the solubility of CO2
7.05
6.8
is reduced, and shows alkaline pH
0 30 60 90 120 150 180 210 240
Time after sampling, min very quickly after thawing.
48
b.Plasma
The figure shows the observation with plasma obtained using EDTA-2Na.
EDTA itself is rather strong acid,
Changes of mouse plasma pH after sampling
but its sodium salt is weakly
8 acidic, and used as anticoagulant.
7.8 So, the plasma showed slightly
7.6 acidic pH at 15 min. after
7.35
7.4
7.35 preparation. pH also went up, but
7.26
pH
7.2
7.17 7.31 7.32 not so markedly, to 7.2 after 1 hour,
7.09 7.25
6.94 7.15 and 7.3 after 4 hours.
7 7.07
6.84
6.91 EDTA-2Nais generally used at
6.8
6.82
final concentration of 1mg/ml,
6.6
0 30 60 90 120 150 180 210 240 namely 0.1%. It reduced plasma
Time after sampling, min
pH to 6.8 at first, and showed
some buffering action to resist pH upraising due to CO2 loss. B B
A long storage in a home freezer is not recommended because the sample is frozen very
slowly and the solute may separate from solvent, and is concentrated at to the bottom of the
storage tube. This may sometimes cause inactivation.
When samples are frozen and stored, first snap-freeze the sample using dry ice-acetone
mixture or dry ice-ethanol, then transfer them into a freezer.
Repeated freezing and thawing is also harmful to the protein, and may cause inactivation.
When samples are taken out from the freezer and thawed, never forget to vortex these
49
samples because the solution after thawing is not homogeneous, and the bottom area contains
more solute.
How many wells should we run per one sample?
– Number of replicate and confidence limit of mean
In ELISA someone assay
samples in duplicates, and
others in triplicates. The
graph shown below indicates
how 95% confidence limits
change according to number of
replicate.
If number of replicate is n,
95% confidence limit of the
mean is calculated as;
Mean ± tn-1(0.05) x SE
B B
50
Examples of guideline for stability test using LQC (low content QC) and HQC ,by triplicates
assay.
Stability at room temperature: 2 hours
Stability at refrigerator temperature: 24 hours
Stability against freezing-thawing: 3 times
Stability for long period: at –20℃ or−80℃
We tested mouse insulin stability in serum.
Serum samples were stored in a refrigerator
(4oC) and in a freezer (-20oC). As shown in
P
P
P
P
51
4. Pipettes
Selection of pipettes
Pipettes for samples and standard solutions are especially important because their
precision directly influence the assay precision. When you add samples or standards at a
volume of 5 or 10 µl, for example, use a pipette with the maximal setting volume of 5 or 10µl,
and never use with the maximum setting volume of 100µl or more. In other words, use
volume-matched pipette. If you measure 100µl with maximum 100µl pipette, the precision of
the delivery volume is about 1%, however, when you measure 5µl with the same pipette, the
precision will be nearly 30%.
With a widely used tip-type pipette, the important factor is the temperature of the solution
to be delivered. If the solution temperature is higher than room temperature, it may cause an
expansion of the air in the tip and the pipette, thus delivers smaller volume of the solution.
On the other hand, colder solution than room temperature causes shrinkage of the air, and
delivered volume will be more than setting. If the sample solution were highly viscous, the
liquid attached to the inner wall of the tip would be more than that of thinner solution, so the
first delivery volume would be less. The amount of solution attached to the outside of the tip
would be also important, and in order to remove this, “touch and go” procedure is necessary.
We do not recommend a multi-channel pipette. We are afraid that it possibly scratches the
bottom of wells because of difficulty of holding it in parallel with wells, resulting in removal
of antibody coating the surface of the bottom and big variation.
In pipetting reagent solution to be added to all the wells, we recommend a repeating
dispenser such as ”Eppendorf multipette plus”. Using this type of pipette, we can dispense
rapidly and are able to save time without scratching the bottom.
Whenever you start using a new type of pipette, please, read the instruction paper from the
pipette maker carefully to avoid misuse of the pipette. Please, refer to Part V.
Contents
Coloration in general
Standard curve
Samples
Assay precision (variation)
Plate seal
Reagents
52
Coloration in general
Troubles Possible Causes and Measures
Poor or no coloration after the 1) The standard or samples might not be added.
last step.
2) Reagents necessary for coloration shown below might not
be added.
①Biotin-labeled antibody
②HRP-conjugated avidin
③TMB or OPD
3) Wrong reagents related to coloration might have been
added.
Wrong dilution of biotin-labeled antibody or HRP-avidin
conjugate.
4) Contamination of enzyme inhibitor(s).
Bottles or vessels used for dilution of HRP-avidin
conjugate might have been contaminated with sodium
azide (NaN3) or fluoride, and inactivates HRP..
B B
Satisfactory absorbance was The plate-reader might not be adjusted to the correct
not obtained by the wavelength (450nm for TMB). ⇒ Check the reader and
densitometer though the adjust the wavelength. In the case of a filter-type
coloration is apparent to the plate-reader, check the wavelength of the filter, and replace
eye. for right one when wrong.
The wells showed coloration The chromogenic substrate TMB used in our assay kits
before the addition of reaction shows bluish color when oxidized by HRP at the neutral pH,
stopper. and turns to yellow-orange color under an acidic pH caused
by addition of sulfuric acid (the reaction stopper). This color
absorbs the light of 450nm wave length. So, the coloration
before the addition of the reaction stopper is natural.
53
Coloration
After enzyme action After reaction-stopper
All the wells showed high TMB solution might have been oxidized by some agents..
coloration.
Standard curve
Troubles Possible Causes and Measures
Flat standard curve. Standard solutions are not added.
All the wells for the standard In the dilution of original standard solution, other solution
curve showed very low
coloration, though sample than the right original standard solution might be used, or
wells gave good coloration. simply the original standard solution was forgotten.
The wells used for blank are in most cases located at A1-A2
or H1-H2 (in duplicated standard wells). And are most liable
54
to be influenced by the edge effect.
One possible cause of the edge effect on such blank wells
may be heat from the fingers which keep holding the
well-plate during the addition of TMB solution. Please, do
not hold the well-plate. To avoid the holding, we recommend
the use of “non-slip sheet”…A very cheap and thin rubber
sheet generally used under a doormat. In our laboratory, we
cut such sheet to a size a little bigger than a well-plate, then
put a plate on it. By doing this the plate will never move
while pipetting without holding.
If you do not like to use non-slip sheet, just hold the plate
at a side with a fingertip to minimize the heat transfer.
55
Washing repetition and blank absorbance
(Mean & SD)
0.180
0.160
Absrobance ⊿450(620) nm
0.140 0.118
0.097
0.120
0.100
0.080
0.060
0.040
0.020
0.000
Washing 4 times Washing 8 times
1
5) Possibility of excessive reaction
Incubation of the well-plate must be carried out at the
right temperature for the right period indicated in the
instruction paper.
The standard curve obtained There might be some mistake in the serial dilution of the
was not smooth. original standard solution. Pay attention to volumes of
standard and buffer solutions and enough mixing.
The standard curve obtained 1) The standard preparation might have been denatured.
moved toward right than that
shown in the instruction 2) The wrong initial dilution of the original standard
paper. solution.
In duplicated standards, those This may due to “edge effect”. Please, read above section
wells located along the edge of describing the edge effect.
the plate always gave higher
absorbance than inner wells.
56
Samples
Troubles Possible Causes and Measures
1) In case NaF-coated blood-sampling tubes are used, or
Assay values were not NaN3 is added to assay sample as a preservative, they may
obtained because samples
B B
57
Lower assay values with 1) The substance in the sample might have been denatured
normal animal samples during the storage.
compared with those obtained
in the previous assays using 2) There might be some seasonal or diurnal changes in the
the same kit. blood level of the substance to be measured.
Higher assay values with 1) This might be due to the same reason stated above, i.e.
normal animal samples difference in the purity of the standard preparation used.
compared with those obtained
with other maker’s kit, or 2) The standard preparation in the present assay might be
those appeared in previous inactivated by denaturation. Please, check the standard
reports. curve if the curve has not shifted to the right.
Assay values were not 1) pH of the sample might be out of the permitted range of
obtained with samples from the assay (pH 6.5 –8.0). Please, check pH and, if necessary,
the tissue or cellar extraction, neutralize by dilution with buffer, or by small volume of
chromatographic, isoelectric acidic or alkaline solution.
focusing, or electrophoresis.
2) Contamination with some organic solvents. Organic
solvents may interfere with the assay reaction. Please, dilute
them with buffer, or remove solvents with nitrogen stream or
placing under reduced pressure for same period.
58
scratching.
The antibody coated on the bottom of well is not so stable,
and may be removed by scratching or hard water jet. The loss
of antibody makes heterogeneity among wells.
3) Assay might be started while the well-plate was still cooler
than room temperature, and this made the heterogeneity in
the temperature among wells, and the edge effect happened.
The well-plate and all reagents as well as assay samples
which have been stored in a refrigerator, should be set back
to room temperature by placing them outside at least for 1.5
hour before starting assay.
4) Air stream, warmer or cooler than room temperature from
air conditioner, cooling system of various apparatus
including personal computer might influence well-plate
temperature. Heat radiation from a stove or instrument also
might give the same effect (edge effect).
5) Air stream from air conditioner or other instruments
might dry wells.
6) Insufficient removal of washing buffer from the wells
might dilute reagent solution added in the following step of
the procedure. The washing buffer should be completely
removed by striking well plate upside down on folded several
sheets of paper towel.
Assay variation due to pipetting
1) Variation of pipetting of standard solutions and samples
directly caused assay variation. To use proper pipette in
proper way is one of the most important points in ELISA.
The upper limit of the pipette must be coordinated to the
volume of standard or sample. Never use 100µl pipette, for
instance, for delivering 5 - 10µl standard or sample solution.
Also, it is necessary to check the variation of every pipette
with proper interval.
2) There are two methods for the tip-type pipette do not mix
up “Pre-wetting method” and “Co-washing method”. These
methods were described in the text (Part V).
3) Temperature of solution to be delivered might influence
variation in assay. If solution to be delivered is cooler or
warmer than room temperature, the volume of the solution
may be smaller or larger than setting, respectively. All the
solutions used in assay should have been stayed at room
temperature for enough period to get to the room
temperature.
4) Choking up of the pipette tip might influence the variation
by decreasing the volume to be delivered. If plasma
samples are frozen-stored and thawed, there formed fibrin
particles which chokes up a pipette tip, interfering with
pipetting. Please, check carefully the state of the samples
after thawing, and if you find fibrin particles, remove them
by any proper way.
59
5) Big variation would be obtained if the sample is not
homogeneous. When serum/plasma samples are stored
frozen, please be careful about the freezing method and
temperature. Slow freezing causes concentration gradient of
protein components, leaving high concentration in the
bottom. Also, when thawing thinner part will remain as ice
at the top. So, samples should be snap-frozen in a very low
temperature obtained by using dry ice-acetone mixture or
dry ice-ethanol mixture, and stored under ultralow
temperature if possible. After thawing, please, stir the
sample well on a vortex mixer.
Plate seal
Trouble Possible Cause and Measure
Plate seal could not be The seal might be removed soon after the plate was taken out
removed completely, and some from a refrigerator.
parts remained on the plate.
The plate seal has been attached to the plate with heat
(without any glue). If you try to remove seal from the plate
soon after taking out from a refrigerator, it is not easy to
remove completely. Please, wait until the plate gets back to
the room temperature. At room temperature, the seal is
easily peeled off.
Reagents
Trouble Possible Cause and Measure
Some crystals were found in From the solubility of the components, some crystals might
the reagent solution. be formed in reagent solution. In such case, please, warm up
the solution by placing the bottle in warm water (lower than
37oC) until those crystals are dissolved, then stir the bottle
P P
Revised 2010/03/25
60